An effective non-viral gene delivery agent must 1) bind DNA, and 2) facilitate various steps necessary for transfection. While much effort has gone into the design of such agents, one basic question remains unclear, namely: how tightly should these compounds bind DNA in order to achieve the maximum efficiency of gene delivery? One possible answer is that the optimal DNA- binding stability of a compound depends on which specific step in delivery it mediates. Thus, I propose to quantitatively and systematically examine how DNA-binding stability affects three crucial steps in gene delivery, namely: cell-specific uptake, endosomal translocation, and nuclear transport. In many medically relevant human cell lines, all of these steps can be mediated by protein sequences - a fact that has led to the development of modular, protein-based delivery systems. I will fuse protein sequences known to catalyze all three of these steps to DNA binding domains derived from two well-characterized repressor proteins, LacR and TetR(D). Both of these proteins bind to their respective operators with a high affinity, and more specifically, with a high degree of stability. The stability of the protein-DNA complexes will then be varied from weak to covalent with little change in overall chemistry by simply modifying their specific DNA binding sites. Furthermore, since small, non-toxic molecules induce the dissociation of Lac and Tet proteins, these proteins will also contain a novel triggered release mechanism. The long-term goal of this project is to create a safe synthetic delivery system with high transfection efficiency, and a useful framework with which other gene delivery agents can be designed.